The biosynthetic pathway known as the shikimate pathway or “common aromatic pathway” leads to the production of many aromatic compounds, including the aromatic amino acids and other compounds such as folate, melanin, indole, catechol, enterochelin, shikimate, dehydroshikimate and L-DOPA. In addition, by introducing specific cloned genes into an organism having the shikimate pathway, the range of compounds that can be produced is greatly expanded. Production of indigo via the aromatic amino acid pathway is an example of the metabolic potential of this pathway.
The cost-effective and efficient biosynthetic production of compounds or derivatives thereof along the common aromatic pathway require that carbon sources such as glucose, lactose and galactose be converted to the desired product with high percentage yield. Thus, from the standpoint of industrial biosynthetic production of aromatic compounds or other biosynthetic derivatives along the common aromatic pathway, it would be valuable to increase the influx of carbon sources into and through the common aromatic pathway, thereby enhancing the biosynthetic production of the desired compound.
Phosphoenolpyruvate (PEP) is one of the major building blocks that cells use in their biosynthetic routes, particularly in amino acid biosynthesis (see FIG. 1). For example, the synthesis of one molecule of chorismate (the common precursor to all of the aromatic amino acids) requires two molecules of PEP. To date, approaches taken to increase the influx of carbon sources into and through the common aromatic pathway typically relate to increasing the PEP supply in the cell by eliminating pyruvate kinase (pyk mutants) [1] and/or eliminating PEP carboxylase (ppc mutants) [2]. A third approach to increasing the PEP supply in the cell is to amplify the expression of the pps gene (encoding PEP synthase, which converts pyruvate to PEP) (U.S. Ser. No. 08/307,371, the disclosure of which is incorporated herein by reference). Additional approaches to increase the flux of carbon into and through the common aromatic pathway relate to increasing the intracellular supply of D-erythrose 4-phosphate (E4P), the other necessary precursor (with PEP) for aromatic biosynthesis. This approach may utilize overexpression of a transketolase gene (tktA or tktB), the product of which (transketolase) catalyzes the conversion of D-fructose 6-phosphate to E4P (U.S. Pat. No. 5,168,056, the disclosure of which is incorporated herein by reference). Another approach to increasing E4P availability may utilize overexpression of the transaldolase gene (talA) which encodes the enzyme transaldolase [3], which catalyzes the conversion of D-sedoheptulose 7-phosphate plus glyceraldehyde 3-phosphate to E4P plus fructose 6-phosphate.
Contrary to the methods previously described, the present invention addresses the issue of increasing PEP availability, and thus carbon flow into a given pathway, by generating strains capable of transporting glucose without consuming PEP during the process. Thus, the conserved PEP is then re-directed into a given metabolic pathway for the enhanced production of a desired product. These strains were generated by inactivating the PEP-dependent phosphotransferase transport system (PTS) utilized by such strains to transport glucose, and then selecting mutants that were capable of transporting glucose efficiently by a non-PTS mechanism (PEP-independent). Using the strategy of inactivating the PTS, the inventors have found that PEP is not consumed in glucose transport and, therefore, can be redirected to other metabolic pathways. These strains (Pts−/glucose+) have successfully been employed to increase production of tryptophan, phenylalanine, tyrosine and other compounds and are contemplated to be useful in producing other aromatic as well as non-aromatic compounds along metabolic pathways in biological systems. For example, oxaloacetate (OAA) is synthesized by at least two routes: (i) through the tricarboxylic acid (TCA) cycle; and (ii) through an anaplerotic route; the latter being catalyzed by PEP carboxylase (PPC) which converts PEP and CO2 to OAA. Elimination of the PTS would increase the level of PEP available to the PPC enzyme, thus enhancing OAA production. Since OAA is the precursor of aspartate, lysine, methionine, isoleucine and threonine (see FIG. 1), production of any of the latter compounds could be enhanced in a Pts−/glucose+ strain.